The invention relates to imaging of gels stained with fluorescent material, in particular to imaging of two-dimensional gels such as protein gels, but also to imaging of one-dimensional gels, such as electrophoresis gels. The invention further relates to excision of gel material on the basis of its fluorescence.
Protein gels are used in various biochemical processes. Proteins are manipulated in one or two dimensions in a gel. For example, manipulation may be in the form of protein migration, where migration rate depends on protein molecule size with the smallest proteins moving fastest. Another kind of manipulation is based on response to an electric field which can be used to force alkali and basic proteins to move in opposite directions with a migration rate that depends on the degree of alkalinity or acidity. So-called two-dimensional gels allow protein migration based on size and acidity to be performed in orthogonal directions in the same gel.
To measure the locations of the proteins in the gel, the proteins are marked with a fluorescent stain. The fluorescence is then measured in an optical device which includes an excitation source and photodetection device. The amount of protein at a particular location is inferred from the strength of the fluorescence measured at that point by the photodetection device.
Once a gel location of interest has been located by the optical device, by virtue of its strong fluorescence, that part of the gel is typically removed and placed in the well of a well plate for further processing. This is done conventionally by transferring the gel dish to a robot fitted with a gel coring head. The gel coring head is fitted with a number of corers, for example 8, which can cut out and suck up circular cross-section plugs of gel and deposit them into a well of a well plate.
A large number of fluorescent stains are known. The stains typically have relatively broadband characteristics with one or more broad absorption or excitation bands and a single strong broad emission band. A widely used fluorescent stain is sypro ruby which has excitation bands in the ultraviolet (UV) and blue and emits in the red, peaked at around 620 nm.
Two kinds of optical device are known for measuring gel fluorescence, as now described with reference to the figures.
FIG. 6 is a schematic side view of an imager in which a charged coupled device (CCD) detector unit 100 comprising a CCD chip 102 and objective 104 is arranged above a shelf 106 on which a gel dish 108 can be placed centrally about the optical axis “O” of the CCD detector unit 100. Alongside the CCD detector unit 100, banks of blue light emitting diodes (LEDs) 110 are arranged facing the center of the shelf where gel dishes are to be placed. The blue LED banks 110 are used to excite fluorescence in the gel which is then measured by the CCD chip. The imager is built into a light-tight housing 112 accessed by a hinged door (not shown). An imager of this kind is the Fuji LAS-1000.
FIG. 7 is a schematic side view of a scanner in which a photomultiplier tube (PMT) 120 and objective 122 are used to measure fluorescence from a gel contained in a gel dish 124 which is excited by raster scanning a 488 nm laser beam 126 generated by an argon ion laser 128 over the gel dish. Raster scanning is achieved by a movable mirror arrangement 130. The scanner is arranged in a light tight housing 132.
It will thus be appreciated that the principal difference between the imager and the scanner is that the imager is a parallel device and the scanner a serial device.
The imager is quicker, but suffers from the limitation that the blanket illumination from the LEDs provides a non-uniform intensity distribution across the gel, which results in errors in the correlation between protein density and fluorescence intensity. This problem is exacerbated by the CCD chip imaging the reflections of individual LEDs themselves as well as the gel.
The scanner provides uniform illumination, since the argon ion laser can be well power stabilized, but is slow. Moreover, use of an argon laser is undesirable since it is a bulky inefficient item that may need air or water cooling and three-phase power supply depending on the optical power required.
As is also apparent, both the scanner and the imager are stand-alone devices. Excision of the gel material identified by the fluorescence analysis needs to be performed in a separate machine, namely a robot with excision capability.